When using SDS to "coat" proteins in negative charge for SDS-PAGE, is a concentration under the critical micelle concentration used? I assume it is (otherwise the proteins would be just brought into the hydrophobic centre of the micelles?).

Generally, moderate concentrations of mild (i.e., nonionic) detergents compromise the integrity of cell membranes, thereby facilitating lysis of cells and extraction of soluble protein, often in native form. Using certain buffer conditions, various detergents effectively penetrate between the membrane bilayers at concentrations sufficient to form mixed micelles with isolated phospholipids and membrane proteins.

Denaturing detergents such as SDS bind to both membrane (hydrophobic)and nonmembrane (water-soluble, hydrophilic) proteins at concentrations below the CMC, i.e. as monomers. The reaction is equilibrium-driven until saturated. Therefore, the free concentration of monomers determines the detergent concentration. SDS binding is cooperative (the binding of one molecule of SDS increases the probability that another molecule of SDS will bind to that protein) and alters most proteins into rigid rods whose length is proportional to molecular weight.

Nondenaturing detergents such as Triton X-100 have rigid and bulky nonpolar heads that do not penetrate into water-soluble proteins; consequently, they generally do not disrupt native interactions and structures of water-soluble proteins and do not have cooperative binding properties. The main effect of nondenaturing detergents is to associate with hydrophobic parts of membrane proteins, thereby conferring miscibility to them.

At concentrations below the CMC, detergent monomers bind to water-soluble proteins. Above the CMC, binding of detergent to proteins competes with the self association of detergent molecules into micelles. Consequently, there is effectively no increase in protein-bound detergent monomers with increasing detergent concentration beyond the CMC.

Detergent monomers solubilize membrane proteins by partitioning into the membrane bilayer. With increasing amounts of detergents, membranes undergo various stages of solubilization. The initial stage is lysis or rupture of the membrane. At detergent:membrane lipid molar ratios of 0.1:1 through 1:1 the lipid bilayer usually remains intact but selective extraction of some membrane proteins occurs. Increasing the ratio to 2:1, solubilization of the membrane occurs resulting in mixed micelles. These include phospholipid-detergent micelles, detergent-protein micelles, and lipid-detergent-protein micelles. At a ratio of 10:1, all native membrane lipid:protein interactions are effectively exchanged for detergent:protein interactions.

The amount of detergent needed for optimal protein extraction depends on the CMC, aggregation number, temperature and nature of the membrane and the detergent. The solubilization buffer should contain sufficient detergent to provide greater than 1 micelle per membrane protein molecule to ensure that individual protein molecules are isolated in separate micelles."